Adenovirus mutants with deleted protease gene

ABSTRACT

An adenovirus vector/packaging cell line system is disclosed, in which the vector replication is blocked by deletion of a single gene, which deletion does not interfere with any other viral functions. The deleted gene is the gene of the adenovirus protease. The protease is expressed in a complementing (packaging) cell line through a regulatable expression cassette which induces no toxic effects in the cells, thus making the generation and propagation of the vector easier and more efficient. As the deleted gene is highly specific of adenovirus, no complementation of the gene in transduced cells is expected, which increases the safety of the new vectors for gene transfer purposes. Also disclosed is a new system of generating recombinant adenovirus vectors by positive selection of recombinants deleted for the endogenous protease gene, which gene is cloned in another region of the adenoviral genome.

FIELD OF THE INVENTION

The present invention relates to gene therapy, in particular torecombinant adenovirus vectors useful for gene transfer and proteinproduction, for applications in gene therapy and functional genomics andvaccination, and to complementing cell lines for the generation andpropagation of such vectors. More particularly, the invention relates toadenovirus mutants deleted at least for the gene of the adenovirusprotease, to complementing cell lines expressing the protease and tocorresponding gene transfer vectors.

BACKGROUND OF THE INVENTION

The term “gene therapy” is usually understood to mean the process inwhich a gene is introduced into the somatic cells of an individual withthe aim of being expressed in the cells, to produce some therapeuticeffect. Initially this principle was applied to cases where anadditional normal copy of a defective gene was provided to restore thesynthesis of a missing protein, such as an enzyme. The concept of genetherapy has since been broadened to include several other approaches. Inparticular, the transferred gene (transgene) may code for a protein thatis not necessarily missing but that may be of therapeutic benefit anddifficult to administer exogenously, for example IL-2 or antitumorcytokines. This form of gene therapy aims to enhance in vivo productionof potentially therapeutic proteins. This approach is similar to genevaccination, where the transferred gene is introduced into the cells toexpress a protein acting as an antigen inducing a protective immuneresponse of the host's immune system. Another form of gene therapyinvolves transferring into cells non-physiological sequences which haveantiviral activity, such as antisense oligonucleotides or sequences.Finally, so-called suicide genes can be transferred into undesirablecells (cancer cells or infected cells), to sensitize them to specificsubstances. When these substances are administered subsequently, theytrigger selective destruction of the targeted cells.

Gene delivery systems which transfer the desired gene into the targetcells are based either on physico-chemical or on biological methods. Ineach case the desired gene can be transferred into cells either invitro, by extracting cells from an organ and reintroducing the cellstransfected in vitro into the same organ or organism, or in vivo, i.e.,directly into an appropriate tissue. Known physico-chemical methods oftransfection include, for example, gene gun (biolistics), in situ nakedDNA injections, complexes of DNA with DEAE-dextran or with nucleicproteins, liposomal DNA preparations, etc. Biological methods,considered to be a more reliable alternative to physico-chemicalmethods, rely on infectious agents as gene transfer vectors. In thisgroup of methods, viruses have become infectious agents of choice, dueto their inherent capability of infecting various cells. The transfer ofa foreign gene by a viral vector is known as transduction of the gene.

Several virus classes have been tested for use as gene transfer vectors,including retroviruses (RSV, HMS, MMS, etc.), herpesviruses (e.g., HSV),poxviruses (vaccinia virus), adenoviruses (Ad, mainly derived from type5 and 2 Ad) and adeno-associated viruses (AAV). Of those,adenovirus-based vectors are presently considered to be among the mostpromising viral vectors, due to their following properties, some ofwhich are unique to this group of vectors: (i) adenovirus vectors do notrequire cell proliferation for expression of adenovirus proteins (i.e.,are effective even in cells at the resting phase); (ii) adenovirusvectors do not integrate their DNA into the chromosomes of the cell, sotheir effect is impermanent and is unlikely to interfere with the cell'snormal functions; (iii) adenovirus vectors can infect non-dividing orterminally differentiated cells, so they are applicable over a widerange of host cells; (iv) adenovirus vectors show a traducing efficiencyof almost 100% in a variety of animal cultured cells and in severalorgans of various species in vivo; (v) adenovirus vectors usuallypossess an ability to replicate to high titer, a feature important forthe preparation of vector stocks suitable for the achievement ofefficient transduction in vivo; (vi) adenovirus vectors can accommodatelarge inserts of exogenous DNA (have a high cloning capacity); (vii)recombination events are rare for adenovirus vectors; (viii) there areno known associations of human malignancies or other serious healthproblems with adenovirus infections; (adenovirus type 5 is originallyknown to cause cold conditions in humans; live adenovirus of that typehaving the ability to replicate has been safely used as a human vaccine(Top et al.,J.I.D.,124,148-154; J.I.D.,124,155-160(1971)).

Structurally, adenoviruses are non-enveloped viruses, consisting of anexternal capsid and an internal core. Over 40 adenovirus subtypes havebeen isolated from humans and over 50 additional subtypes from othermammals and birds. All adenoviruses are morphologically and structurallysimilar, even though they differ in some properties. Subtypes of humanadenoviruses are designated according to serological response toinfection. Of those, serotypes Ad2 and Ad5 have been studied mostintensively, and used for gene transfer purposes since the 80s.Genetically, adenovirus is a double-stranded DNA virus with a lineargenome of about 36 kb. The genome is classified into early (E1-E4) andlate (L1-L5) transcriptional regions (units). This classification isbased on two temporal classes of viral proteins expressed during theearly (E) and late (L) phases of virus replication, with viral DNAreplication separating the two phases.

A viral gene transfer vector is a recombinant virus, usually a virushaving a part of its genome deleted and replaced with an expressioncassette to be transferred into the host cell. Additionally to a foreign(exogenous) gene, the expression cassette comprises components necessaryfor a proper expression of the foreign gene. It contains at least apromoter sequence and a polyadenylation signal before and after the geneto be expressed. Other sequences necessary to regulate or enhance thegene expression can be included in the cassette for specificapplications.

The deletion of some parts of the viral genome may render the virusreplication-incompetent, i.e., unable to multiply in the infected hostcells. This highly desirable safety feature of viral vectors preventsthe spread of the vector containing the recombinant material to theenvironment and protects the patient from an unintended viral infectionand its pathological consequences. The replication-defective virusrequires for its propagation either a complementing cell line (packagingcell line) or the presence of a helper virus, either of which serves toreplace (restore in trans) the functions of the deleted part or parts ofthe viral genome. As it has been shown that the production ofrecombinant viral vectors free of replication-competent helper virus isdifficult to achieve, the use of packaging cell lines for thepropagation of replication-incompetent viral vectors is considered to bethe best choice for gene therapy purposes.

Early adenovirus vectors (sometimes referred to as first generationadenovirus vectors, or singly deficient vectors) relied on deletions(and insertions) in coding regions E1 and/or E3 of the viral genome(see, for example, U.S. Pat. Nos. 5,670,488; 5,698,202; 5,731,172). E3deletion was usually performed to provide the necessary space for theinsertion of foreign genes of a limited size. The E3 region isnon-essential for virus growth in tissue culture, so that vectorsdeleted only in E3 region could be propagated in non-complementingcells. As E1 region is essential for the virus growth, E1-deletedvectors could only be propagated in complementing cells, such as human293 cells (ATCC CRL 1573), a human embryonic kidney cell line containingthe E1 region of human Ad5 DNA.

One of critical issues in the development of safe viral vectors is toprevent the generation of replication-competent virus during vectorproduction in a packaging cell line or during the gene therapytreatment. This may happen as a result of a recombination event betweenthe genome of the vector and that of the packaging cells, or of thevector and the wild-type virus present in the recipient cells of thepatient or introduced as a contaminant in the process of producing therecombinant virus. On occasion, a recombination event could generate areplication-competent virus carrying the transgene, which virus mightspread to the environment. Even though recombination events are rare forE1-deleted adenovirus vectors, their in vivo replication and the ensuingrisks could not be completely prevented, and generation ofreplication-competent adenovirus was demonstrated during the preparationof viral stocks. Another danger is the loss of replication deficiency(and the return to a phenotypic state of multiplication) throughcomplementation in trans in some cells which produce proteins capable ofreplacing proteins encoded by the deleted regions of the viral genome.This was demonstrated for E1-deleted adenoviruses.

Attempts to improve the safety and cloning capacity of adenovirusvectors resulted in development of a new generation of multiplydeficient adenovirus vectors (also referred to as second generation ormultiply deleted vectors). Additionally to deletions in E1 and/or E3coding regions, these vectors are also deleted in other regions of theviral genome essential for virus replication, such as early regions E2and/or E4 (see, for example, WO 95/34671; U.S. Pat. No. 5,700,470; WO94/28152) or late regions L1-L5 (see, for example, WO 95/02697). Otherknown approaches to improve the safety of adenovirus vectors include,for example, relocation of protein IX gene in E1-deleted adenovirus(U.S. Pat. No. 5,707,618) and inactivation of the gene IVa2 in amultiply deleted adenovirus (WO 96/10088). All second generationadenovirus vectors are replication-deficient and require complementingcell lines for their propagation, to restore in trans the deleted orinactivated functions of the viral genome. More importantly, suchvectors show an improved resistance to recombination when propagated incomplementing cell lines or transferred into recipient cells of apatient, making recombination events virtually nonexistent and improvingthe safety of gene therapy treatments.

Even though adenovirus vectors with improved resistance to recombinationare known in the prior art, as exemplified above, they are not equal interms of their gene transfer efficiency, cloning capacity, toxicity tohost cells, severity of immune response induced in patient's organism,ease of propagation (which may be limited by toxicity of viral gene(s)to packaging cell lines), ease of generation (which may be limited by anexpression cassette harboring toxic genes), and in vivo regulatabilityof the exogenous (foreign) gene expression. Consequently, there exists acontinuous need for adenoviral vectors having at least some of theseproperties improved. The present invention provides new adenoviralvectors and complementing cell lines for their generation andpropagation, which are free of many prior art limitations.

Adenoviral vectors may also be useful as tools for the production ofproteins, for example for the purpose of functional genomics studies inmammalian cells. Cloning and expressing numerous genes allows thegeneration of mini-libraries useful for various applications, such assignal transduction studies or screening antisense DNA constructs. Thisapplication of adenoviral vectors requires a cloning system in whichgeneration and selection of recombinant mutants can be easily performed.The present invention provides such a novel system of cloning DNAsequences using adenoviral vectors.

SUMMARY OF THE INVENTION

The present invention provides an adenovirus vector/packaging cell linesystem in which the vector replication is blocked by deletion of asingle gene, not a viral region, which deletion does not interfere withany other viral functions. The deleted gene is the gene of theadenovirus protease. The protease encoded by the deleted gene isexpressed in a complementing (packaging) cell line through a regulatableexpression cassette which induces no toxic effects in the cells, thusmaking the generation and production of the vector easier and efficient.As the deleted gene is highly specific of adenovirus, no complementationof the gene in transduced cells is to be expected, which increases thesafety and suitability of the protease gene deleted vectors for genetransfer purposes.

When additionally deleted for E1 region of adenoviral genome, thevectors of the invention are blocked for replication, but are capable ofa single round of replication if deleted only for the protease gene. Thelatter feature permits an enhanced expression of the transgene intransduced cells, which may be of importance in some applications, forexample to achieve localized enhanced expressions of transgenes (in situtumor therapy) or efficient vaccinations without boosting.

The invention further allows positive selection of E1-deleted,protease-deleted recombinant adenovirus vectors by providing theprotease gene as part of an expression cassette inserted in place of theE1 region in a shuttle vector. In vivo recombination of the shuttlevector with a protease-deleted adenoviral genome generates viablerecombinants only when rescuing the protease cloned in E1 region. Nonrecombinant viral genomes are not able to grow due to the proteasedeletion, ensuring that only recombinant viral plaques are generated.

Consequently, it is an object of the present invention to provide novelcell lines capable of hosting an adenovirus mutant deleted for theprotease gene, which cell lines contain DNA expressing the adenovirusprotease.

It is a further object of the present invention to provide a method forproducing novel cell lines capable of hosting an adenovirus mutantdeleted for the protease gene, which cell lines contain DNA expressingthe adenovirus protease.

It is a further object of the present invention to provide a method ofusing cell lines capable of hosting an adenovirus mutant deleted for theprotease gene and containing DNA expressing the adenovirus protease togenerate and propagate adenovirus mutants deficient for the adenovirusprotease gene.

It is a further object of the present invention to provide noveladenovirus mutants deleted for the adenovirus protease gene.

It is a further object of the present invention to provide noveladenovirus mutants deleted for the protease gene and at least oneadditional adenovirus gene or genomic region.

It is a further object of the present invention to provide noveladenovirus vectors for gene transfer, protein production, gene therapyand vaccination, said vectors deficient at least for the adenovirusprotease gene and containing at least one exogenous gene to betransferred to and expressed in a host cell.

It is a further object of the present invention to provide a novel meansof generating recombinant adenovirus vectors by positive selection ofrecombinants deleted for the endogenous protease, in which the proteasegene is rescued by cloning the gene in another region of the adenoviralgenome.

According to one aspect of the present invention, novel cell lines havebeen generated which are capable of expressing the Ad2 protease genefrom a dicistronic expression cassette, under control of a tetracyclineinducible promoter. The protease is expressed in these cells togetherwith the green fluorescent protein (GFP), the latter used to facilitatecell cloning and expression monitoring. The novel cell lines have beenprepared by transfecting derivatives of 293 cells with pieces of DNAencoding the Ad2 protease and GFP, selecting cells harboring thesepieces (cells expressing the GFP) and amplifying them. The novel celllines, stably expressing the Ad2 protease, produce amounts of proteaseequal to or greater than those reached after comparable infections byadenovirus. The biological activity of the novel cell lines has beendemonstrated by their ability to fully support the reproduction ofAd2ts1 mutant, a temperature-sensitive mutant expressing a functionallydefective protease and to restore normal yields of replication of twonovel adenovirus mutants in which the protease gene has been deleted.

According to another aspect of the present invention, novel mutants ofAd5 deleted at least for the adenovirus protease gene have beengenerated. These novel mutants have been successfully propagated in thecell lines of the invention.

According to yet another aspect, the present invention provides a methodof generating protease-deleted adenovirus mutants having an exogenousgene inserted in E1 coding region, using positive selection of mutantsobtained by in vivo recombination of adenoviral genome deleted forendogenous protease gene and in E1 coding region with a fragment ofadenoviral genome expressing the adenoviral protease gene from anexpression cassette replacing the E1 coding region.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a photograph showing expression of the protease by someclones of 293-tTA-PS and 293-rtTA-PS cells. Total protein extracts (30μg) from different cell lines before (−) and after (+) induction weresubmitted to 14% SDS PAGE and transferred to nitrocellulose sheet. E.coli expressed Ad2 protease (lane 1: E. coli), adenovirus endogenousprotease (lane 3: AdV) and non-transformed cells (lane 2: mock) wereincluded as controls.

FIG. 1B is a photograph showing immunoblot of protein extracts of FIG.1A. Proteins were revealed with an antiactin antibody to check that thesame amount of protein was loaded per well.

FIG. 2 is a schematic representation of all molecular clonings performedto generate bacterial plasmids harboring protease deleted adenovirusregions. A PCR engineered protease deletion was introduced (aftersequencing of the corresponding region) into pDE3 plasmid in which a2378 bp upstream extension has been previously inserted by cloning ofthe RsrII/XhoI 6145 bp fragment from Ad5 genome.

FIG. 3 is a schematic representation of bacterial plasmids harboringprotease deleted adenovirus regions and of the recombinations performedin E. coli to generate bacterial plasmids harboring protease deletedadenovirus genomes. The NdeI/XhoI fragment from pDE3-ext-ΔPS plasmid wasintroduced by homologous recombination in E. coli with eitherpAdEasy1-βgal-GFP plasmid (which harbors an E1/E3 deleted Ad5 genomewith reporter genes βgal of E. coli and GFP) or pTG3026 plasmid (whichharbors an intact Ad5 genome) SgfI digested.

FIG. 4 is a graph showing the effects of induction of proteaseexpression on viability of cells of 293-PS cell lines. Six 6 cm Petridishes were seeded with 2×10⁵ cells of 293 and 293-PS tTA. Aliquots wereexamined for living/dead cells by trypan blue staining on day 0 (D0)through day 5 (D5). Results of overall cell growth of a typicalexperiment are plotted as the count of total living cells as a functionof time (in days) for 293-tTA-PS cells, either induced (I) or notinduced (NI), with 293-tTA as controls.

FIG. 5 is a schematic representation of the molecular cloning performedto generate recombinant adenoviral vectors by positive selection with Adprotease. The recombinant represented here featured an E1-deletion. Ashuttle vector, containing adenovirus 59.4 to 15.5 mu part of the genometo allow recombination, also harbored a triple expression cassettecontaining, among others, the protease gene and a foreign gene ofinterest (X) in place of the E1 region. After linearisation, the shuttlevector was cotransfected in a 293-derived cell line with aprotease-deleted adenovirus genome cleaved in E1. Due to proteasedeletion, only genomes for which recombination, and thus the rescue ofthe protease gene, has occurred, produced viral plaques. The resultingrecombinant viruses harbored no protease gene in L3 region, but theE1-cloned gene and the protease are ectopically expressed from the E1region.

FIG. 6 is a photograph showing a Coomassie blue stained geldemonstrating the ability of Ad5-ΔPS mutant to perform a single round ofreplication in non-complementing cells. A549 cells were inoculated at aMOI of 5 pfu with indicated mutants. 3 days later the cells were lysedin Laemmli buffer. 20 micrograms of protein extracts were loaded perwell and migrated in a 12% acrylamide:bisacrylamide gel. Comparison ofviral protein synthesized by the different mutants (i.e. hexon, 100K)shows that only Ad5-ΔPS mutant produces them in amounts similar to thatof wild-type virus. This confirms the ability of this mutant to performa single round of replication in non-complementing cells.

FIG. 7 is a graph showing the viral yields of different adenoviralmutants in A549 cells. The ability of Ad5-ΔPS mutant to perform a singleround of replication in non-complementing cells was further determinedby titration of the same extracts as presented in FIG. 6. A549 cellswere inoculated at a MOI of 5 pfu with indicated mutants. 3 days later,cells were harvested and extracts were titrated in 293rtTA.PS.7. Titersare indicated in log values (0-9).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As used herein, the term “adenovirus” (Ad) means any adenovirus ofhuman, mammalian, or avian origin (Mastadenovirus, Aviadenovirusfamilies). Of those, human adenoviruses Ad2 and Ad5 are preferred, Ad5being particularly preferred.

In the context of the present invention, the term “adenovirus protease”designates the protease of any adenovirus of human, mammalian, or avianorigin, including analogues, homologues, mutants and isomers of suchprotease. The term “adenovirus protease gene” means the protease gene ofany adenovirus of human, mammalian, or avian origin, includinganalogues, homologues, mutants and isomers of such gene. Even thoughminor differences exist between proteases of different adenoviruses,these proteases are interchangeable. Proteases of human adenoviruses Ad2and Ad5 are preferred, the Ad2 protease being particularly preferred.

The adenovirus protease, first identified by studies on the Ad2ts1temperature sensitive mutant (Weber, J. Virol, 17, 462-471 (1976);Yeh-Kai et al., J. Mol. Biol., 167, 217-222 (1983)), is a key enzyme inthe adenovirus life cycle, serving for maturation of several proteins.Proteins cleaved by this enzyme are the pre-terminal protein (pTP), pVI,pVII, pVIII, pIIIa and the 11K DNA binding proteins (Anderson et al., J.Virol., 12, 241-252, (1973); Boudin et al., Virology, 101, 144-156(1980); Tremblay et al., Biochim. et Biophys. Act., 743, 239-245(1983)). In addition to those substrates, the cleavage of viral 52Kprotein (Hasson et al., J. Virol., 66, 6133-6142 (1992)) and of cellularcytokeratin 18 (Chen et al., J. Virol., 67, 3507-3514 (1993)) play animportant role in the viral cycle. The adenovirus protease is thereforean essential element for virus proteins maturation, for virus entry intohost cells (Cotten et al., Virology, 213, 494-502, (1995); Greber etal., EMBO J., 15, 1766-1777 (1996)) and for release of virions frominfected cells (Chen et al., supra).

Adenovirus protease deleted mutants provide numerous advantages for genetherapy and vaccination. Such mutants, whether deleted or not foradditional genes (e.g., in the E1 coding region), are completelyreplication-deficient. Even though capable of cleaving some cellularproteins, the adenovirus protease is highly specific, making itextremely unlikely that the protease defect in the mutant could beovercome in a mammalian cell, an effect demonstrated for E1 deletedadenovirus mutants in some mammalian cells (Hela and teratocarcinomastem F9 cells: Imperiale et al., Mol. Cell. Biol., 4, 867-874 (1984),Nevins et al., Curr. Top. Microbiol. Immunol., 113, 15-19 (1984);embryonic carcinoma (EC): Keaveney et al., Nature, 365, 562-566 (1993)).This provides an increased safety level for therapeutic applications.For gene therapy applications, a complete blockage of replication ofadenovirus can be reached by deleting the protease gene together withother gene or genes essential to the virus growth, such as E1 codingregion. Recombinant vectors deleted only for the protease and thuscapable of a single round of replication provide interesting vectors forvaccination.

Adenoviruses deleted for the protease gene require for their propagationa cell line capable of providing the protease gene product in trans,such as the cell lines of the present invention. According to onepreferred embodiment, 293S cell lines stably expressing the Ad2 protease(293-PS cells) have been generated. 293S cells were chosen for tworeasons. Firstly, 293 cells allow the propagation of adenovirusessimultaneously deleted in E1 and/or E3 coding region, such asrecombinant adenovirus vectors for gene therapy applications. Secondly,the non-adherent phenotype of 293S cells is advantageous for a scale-upof preparation of deleted adenovirus (Garnier et al., Cytotechnology,15, 145-155 (1994)), for example for the production of vector stocks. Itwould be apparent to those skilled in the art that other cell linescapable of hosting adenoviruses, such as A549, 911, or BMAdE1 would bealso suitable for generating cell lines expressing the adenovirusprotease gene.

According to the preferred embodiment of the invention, induciblepromoters were chosen to achieve regulatable expression of the proteasegene in the cell lines of the invention, namely the tTA and rtTA systems(Gossen et al., Proc. Natl. Acad. Sci. USA, 89, 5547-5551 (1992); Gossenet al., Science, 268, 1766-1769 (1995)). These systems allow forinducible expression of the gene, either by adding or withdrawingtetracycline to the cells. Regulatable expression cassettes were chosenbecause of the ability of the adenovirus protease to disrupt somecomponents of the cellular cytokeratin network (Chen et al., supra).This function appears to play a key role in the phenotypiccharacteristics of adenovirus cytopathic effect, and thus may be atleast deleterious for the host cells. A regulatable expression cassettemakes it possible to limit the expression of the protease, at least at ahigh level, only to periods of time when the inducer is either added orwithdrawn, so that the toxic effect of protease which could hamper thegeneration or propagation of protease-deleted adenovirus is eliminated.(Cells transfected by foreign plasmid DNA are stressed by transfectionand much more sensitive to any toxic effects.)

For recombinant adenovirus vectors for gene therapy and vaccination,putting the transgene into a similar regulatable expression cassetteprovides a number of advantages. By choosing, for example, either tTA orrtTA regulation system, this allows the control of expression of thetransgene either by administering tetracycline or by withdrawing itsadministration, respectively. This can be useful, for example, invaccination of animals for which tetracycline is added on a regularbasis to feeding. Expression of the gene of interest can be in this caseinduced by withdrawing the administration of tetracycline during anappropriate period of time. It would be apparent to those skilled in theart that other regulatable promoters, such as ecdysone or corticosteroidresponsive systems could be used for the practice of the invention.

The cell lines of the invention can be prepared by methods known tothose skilled in the art, in particular by cotransfection of cellscapable of hosting adenovirus with pieces of DNA encoding the adenovirusprotease and pieces of DNA encoding a selection factor, incubating thecells, selecting cells expressing the selection factor, and amplifyingthose expressing the adenovirus protease. The selection factor can beanything which will allow the selection of a cell, such as, for example,an antibiotic resistance protein.

According to the preferred embodiment, the novel complementing celllines of the invention were produced by cotransfecting 293-tTA or293-rtTA cells with plasmid pTR5/PS-DC/GFP (which contains atetracycline regulatable (TR) promoter in a dicistronic cassette (DC)with the GFP and the protease (PS) gene) and with plasmid pTKNeo(comprising the gene of resistance to geneticin (antibiotic G418)) orwith plasmid p3′SS (comprising the gene of resistance to hygromycin),respectively, and selecting transfected cells with these antibiotics.Antibiotic-resistant colonies expressing the GFP protein were amplifiedand several of them selected for further analysis.

To facilitate the screening of recombinant clones, the adenovirusprotease gene was expressed from a dicistronic cassette (Mosser et al.,Biotechniques, 22, 150-161 (1997)) together with a reporter gene ofAquorea victoria green fluorescent protein (GFP) (Prasher et al., Gene,111, 229-233 (1992); Heim et al., Nature, 373, 663-664 (1995)). Afterthe first selection with an antibiotic (G418 or hygromycin), cellsexpressing GFP were selected for further studies by automatedfluorescent cell sorting. This system allowed an efficient generation of293 cell lines stably expressing the active Ad2 protease.

It will be apparent to those skilled in the art that the pieces of DNAencoding the adenovirus protease may be introduced into the cells usingany DNA intracellular delivery system, such as, for example, recombinantplasmids, and by means of any transfection technique, such as calciumphosphate precipitation or liposome technology. Also, cells harboringpieces of DNA encoding the adenovirus protease may be made selectableusing any suitable selection factor, such as the gene of resistance toan antibiotic, which gene can be transfected into the cells by asuitable recombinant plasmid.

To study the biological activity of the recombinant protein,complementation of the Ad2ts1 mutant (Weber, J. Virol., 17, 462-471(1976)) was examined. This mutant encodes a modified P137L proteasewhich is active at the permissive temperature (33° C.) and functionallydefective at 39° C. Replication of the Ad2ts1 on 293tTA-PS and293-rtTA-PS cell lines allowed for restoration of yields similar to thatof the wild-type virus. It was also shown that expression of theprotease was not toxic to the cells but rather slightly impaired thenormal cell growth. The novel cell lines were also shown to restorereplication of two novel adenovirus mutants in which the protease genehas been deleted.

The novel adenovirus mutants deleted for the protease gene can beprepared by methods known to those skilled in the art. In general, thepreparation of a virus mutant relies on preparing first the completegenome of the mutant by joining suitable pieces of DNA, either byligation in vitro or by recombination in a cell. In the latter case,several (usually two or three) fragments of adenoviral DNA containingregions of similarity (or overlap) are transfected into host cells,where they become recombined into a full-length viral genome. Thefragments to be ligated or recombined may contain deletions andmodifications with respect to the wild type viral genome, but mustotherwise contain its entire length. The DNA of the recombinant virus soprepared is then transfected into suitable complementing cells capableof providing in trans viral functions missing from the transfectedrecombinant viral genome as a result of the deletions and modificationsintroduced into the wild type genome. The recombinant virus willmultiply in these cells from which it can be subsequently released, forexample by subjecting cells to several freeze-thaw cycles. Numerousvariations of this general procedure are possible, as would be apparentto those skilled in the art.

According to the preferred embodiment, two novel Ad5 mutants (designatedas Ad5CMVLacZ-CMVGFP-ΔPS and Ad5-ΔPS, respectively) have been generatedaccording to the general procedure outlined above. This was achieved bya series of clonings into bacterial plasmids, followed by recombinationof suitable fragments of the viral genome performed in E. coli, togenerate bacterial plasmids harboring protease deleted adenovirusgenomes. This procedure is summarized in FIG. 2 and FIG. 3 and discussedin more detail in the following Examples.

Ad5-ΔPS mutant is deleted for the protease gene only.Ad5CMVLacZ-CMVGFP-ΔPS is deleted for the protease gene, but also in E1and E3 coding regions of the Ad5 genome. Both mutants have beensuccessfully propagated in the novel complementing cell lines of theinvention expressing the Ad2 protease. Ad5CMVLacZ-CMVGFP-ΔPS mutantcontains in its genome two exogenous genes (transgenes): the gene of E.coli β galactosidase (βgal) and the gene of Aquorea victoria greenfluorescent protein (GFP). These reporter genes can be easily replacedwith genes of therapeutic interest by methods known to those skilled inthe art. In both mutants genes of therapeutic interest can be easilyintroduced by recombination, as both were cloned in bacterial plasmids.

The invention also allows an easy generation of E1-deleted, proteasedeleted recombinant vectors, comprising an exogenous gene or genes(transgenes) in E1 coding region, by providing the protease gene(together with exogenous gene or genes) as part of a di- or tricistroniccassette in place of E1 coding region in a shuttle vector. In vivorecombination of the shuttle vector with a protease-deleted adenoviralgenome generates viable recombinants only when rescuing the proteasegene cloned in E1 coding region. Non-recombinant adenoviral genoms areunable to grow due to protease deletion. This positive selection ensuresthat only recombinant vectors will be generated.

The invention also allows the safe generation of adeno-associated virusvectors due to the total block of replication of the protease deletedmutants that can be used as helpers for this application.

Experimental

The cell lines and vectors of the present invention have been preparedusing techniques well known to those skilled in the art. The followingexamples are provided for better illustration of the invention.

Materials and Methods

Cells and Viruses

293 cells are human embryonic kidney cells expressing high levels of theadenovirus 5 E1A and E1B products (Graham et al., J. Gen. Virol., 36,59-72 (1977)). 293S cells, a non-adherent 293 cells clone has beenpreviously described (Garnier et al., Cytotechnology, 15, 145-155(1994); Massie et al., Bio/Technology, 13, 602-608 (1995)). 293-tTA cellline was described by Massie et al., J. Virol, 72, 2289-2296 (1998), andthe 293-rtTA cell line was obtained in a similar way. Adenovirus Ad2ts1mutant was previously described (Weber, J. Virol., 17, 462-471 (1976)).Adenovirus dl309 is a fully replicative mutant and was previouslydescribed (Jones et. al., Cell, 13, 181-188 (1978)). AdCMV5-GFP is arecombinant adenovirus in which E1 region has been replaced by a CMVdriven GFP expression cassette (Massie et al., Cytotechnology, in press(1999)).

Plasmids

Plasmid pTKNeo was generated by auto-ligation of the BstEII fragment ofpREP 9 (Invitrogen). Plasmid pTR-DC/GFP was previously described (Mosseret al, 1997). This plasmid has been modified from pUHD10.3 (Resnitsky etal., Mol. Cell. Biol., 14, 1669-1679 (1994)) which contains thetTA-responsive promoter with a dicistronic expression cassette.Dicistronic expression is permitted by the encephalomyocarditis virusIRES (Ghattas et al., Mol. Cell. Biol., 11, 5848-5859 (1991)). Theoriginal pTR-DC/GFP was modified by insertion of a Bg1II site. Proteasegene was excised from pAdBM5-PS, by BamHI digestion, sequenced andsubcloned into the Bg1II site of pTR-DC/GFP. Final plasmid,pTR5/PS-DC/GFP thus co-expresses inducibly GFP S65T mutant and Ad2protease genes. Expression of GFP and protease were assayed bytransfection in 293 cells. The transient expression of the protease wasestablished by Western-blot with an anti-protease polyclonal antiserumraised in rabbit with a recombinant protein (from Dr J. Weber,University of Sherbrooke). The expressed protein had the same molecularweight as the native protein from wild-type adenovirus, and wasexpressed only when induced. Plasmid pDE3 was a gift of Dr Lochmüller(Montreal Neurological Institute). This plasmid contains the right endof Ad5 genome from the BamHI site (21562) to the end of the genome, withan E3 deletion. This deletion corresponds to the one described by Bettet al. (1994) and originates from plasmid pBHG11 (extent of thedeletion: 27865-30995). Plasmid pAdEasy-1-βGal-GFP was a gift of Dr He(John Hopkins University, Baltimore, Md.) and has been already described(He et al, 1998). Plasmid pTG3602 (Chartier et al., 1996) was a gift ofDr Mehtali (Transgene SA, Strasbourg, France). Recombinant adenovirusconstruction in E. coli was performed as described respectively by He etal (1998) and Chartier et al. (1996).

Generation of Protease Expressing Cell Lines

293-tTA cell lines were generated by co-transfection of pTR5/PS-DC/GFPand pTKNeo. 293 rtTA-PS clones were generated in a similar way byco-transfecting the same plasmid with the p3'SS (Stratagene) in 293SrtTA. Transfections were achieved by the optimized calcium-phosphateprecipitation method (Jordan et al, Nucleic Acids Res., 15, 24(4):596-601(1996)). For tTA and rtTA, selection drugs were respectively G418and hygromycin (Sigma Chemical).

Selection of Recombinant Cell Clones

After co-transfection and selection, clones of 293S cells expressing theGFP from the dicistronic cassette were selected by screening for theexpression of the GFP by flow cytometry analysis and cell sorting. Flowcytometry was performed using an EPICS Profile II (Coulter, Hialeah,Fla., USA) with a 15 mW argon-ion laser. Cell sorting was carried out onan EPICS V (Model 752, Coulter) multiparameter laser flow cytometer andcell sorter, using the Auto-clone (multiwell automated cell deposition)system. Before selection and sorting, expression of both GFP andprotease was induced by addition (rtTA) or suppression (tTA) ofdoxycycline. For the analysis of GFP expression, cells were sterilycollected and concentrated (1×10⁶ cells/ml) in phosphate-buffered saline(PBS) by centrifugation. The mostly fluorescent cells were gated anddistributed clonally in 96-well plates.

Analysis of Recombinant Protein Expression

Expression of the GFP was checked periodically by flow cytometryanalysis, while expression of the protease was assayed bywestern-blotting. Cells were washed in PBS, centrifuged and frozen.Lysis was carried out in 100 mM Tris-HCl [pH 6.9], 10% glycerol, 2% SDS,and high molecular weight DNA was disrupted by sonication. Prior toassay, total protein contents of extracts were titrated using the DCProtein Assay Kit (Biorad). For electrophoresis, samples were diluted inLaemmli buffer (Laemmli et al., J.Mol.Biol.,88, 749-165, (1974)) andboiled for 5 min. An estimated 20 μg total protein quantity was loadedper well in 14% acrylamide:bisacrylamide (30:1) gels. Afterelectrophoresis, proteins were transferred to nitrocellulose membraneswhich were subsequently blocked overnight at 4° C. with PBS containing5% nonfat dry milk, 0.1% Tween 20. The rabbit anti-protease antibody wasdiluted 1:20000 in the same buffer but with 0.2% Tween 20. As aninternal control, an anti-actin monoclonal antibody diluted 1:10000 wasused. Incubation was overnight at 4° C. Conjugates were used at a1:10000 dilution in the same buffer for 1 hr at room temperature.Revelation was carried out using the ECL chemiluminescence kit(Amersham) according to the manufacturer's instructions.

EXAMPLES

Generation and Isolation of 293 Cell Lines Transformed with Ad2 ProteaseGene

Cell lines were generated by co-transfection and selection withappropriate agents as summarized in Table 1.

TABLE 1 Analysis of the clones obtained from transformation of 293 cellswith protease Selected Plasmids used Selection Clones Clones PositiveCells for transfection agent obtained analyzed clones 293 pTR5/PS-DC/G418 >50 17 7 rTA GFP + pTKNeo 293 pTR5/PS-DC/ hygromycin >50 14 9 rtTAGFP + p3′SS

293 tTA cells were co-transfected with pTR5/PS-DC/GFP and pTKNeo, while293 rtTA were co-transfected with the same plasmid and p3′SS. After a 48hour recovery, transfected cells were submitted to a three weeksselection by either G418 (500 μg/ml) for 293 tTA or hygromycin (150μg/ml) for 293 rtTA. During this time, fresh medium and drug wereapplied to cells twice a week. Throughout the selection process, GFPexpression was monitored on aliquots by flow cytometry analysis. Cellswere then sorted using the multiwell automated cell deposition systemand clonal distribution was visually checked. Expression was thenassessed and only homogenous clones (as checked by unicity of the peakof fluorescent cells) were selected. Results of GFP expression of stableclones are summarized in Table 2.

TABLE 2 GFP expression and induction efficiency in 293-tTA-PS and293-rtTA-PS FI Induction Clone OFF ON Factor 293-tTA-PS-2 138 2838 20293-tTA-PS-11 63 1322 21 293-tTA-PS-15 30 3186 106 293-rtTA-PS-7 35 122835 293-rtTA-PS-10 107 720 7 293-rtTA-PS-17 118 1654 14

Selected cell line clones were tested for the expression of the GFP(basal and induced) by flow cytometry analysis. FI: fluorescence indexcalculated as the percentage of cells expressing GFP by the meanfluorescence value; OFF: GFP expression without induction (50 ngdoxycline per ml for tTA); ON: GFP expression after induction (1 μg perml for rtTA). Induction factor was calculated as the ratio between theFI of the ON state and the FI of the OFF state.

Of all the tested clones, three of 293S-tTA-PS and three of 293S-rtTA-PSclones were selected. Induction efficiency was measured by comparingproducts of the mean fluorescence of one cell by the percentage offluorescent cells (fluorescence indexes: FI). Induction factors rangedfrom 7 to 106 which is in the range of what is usually observed withtetracycline-regulated expression cassettes.

Protease Expression in 293-tTA-PS and 293-rtTA-PS

Of the clones tested for protease expression, three clones of 293-tTA-PSand of 293-rtTA-PS are presented (FIG. 1A). Expression was revealed witha polyclonal rabbit anti-serum raised to the E. coli-expressed protein.Results demonstrate that the expressed protein displays the sameelectrophoretic pattern than that of the endogenous adenovirus protein,and that the expression depends on the induction in all tested clones.The latter assertion was checked by the internal control (cellularactin) which demonstrates that the same amount of protein extract hasbeen loaded in each well. Testing of all obtained clones of rtTA did notallow for observation of higher levels of expression than that reachedwith tTA clones. Level of expression of protease in all selected cloneswere equal or higher than that of the native adenovirus protease (FIG.1A, lane 3). To check that, 293 cells were infected at a MOI of 10 pfuwith AdCMV5-GFP (Massie et al, Cytotechnology, in press (1999)) andprotein extracts were prepared at 48 hrs p.i. Similar level ofexpression was achieved with the clones derived from 293-tTA and293-rtTA cells.

Biological Activity of Protease Expressed by Cell Lines

To study the biological activity of Ad2 protease in transformed celllines, complementation of the temperature-sensitive Ad2ts1 and of twonovel protease deleted mutants by the cell lines was examined. Ad2ts1viral particles produced at 39° C. contain a functionally deficientprotease, and they were used to assess complementation. Results ofone-step growth curves in 293 and 293-PS cell lines (tTA and rtTA) forAd2ts1 are summarized in Table 3 and for both novel protease deletedmutants in Table 4.

TABLE 3 Yield of dl309 and Ad2ts1 from One-Step growth curves indifferent 293-derived cell lines Virus Cell line Temperature Virus titerdl309 293-tTA 33 1.7 × 10⁸ 39 1.2 × 10⁸ 293-tTA-PS-15 NI 33 2.0 × 10⁸ 391.2 × 10⁸ 293-tTA-PS-15 I 33 8.1 × 10⁷ 39 7.0 × 10⁷ 293-rtTA 33 1.6 ×10⁸ 39 1.5 × 10⁸ 293-rtTA-PS-7 NI 33 1.5 × 10⁸ 39 1.0 × 10⁸293-rtTA-PS-7 I 33 7.8 × 10⁷ 39 7.2 × 10⁷ Ad2ts1 293-tTA 33 2.5 × 10⁸ 395.0 × 10³ 293-tTA-PS-15 NI 33 1.6 × 10⁸ 39 9.0 × 10⁷ 293-tTA-PS-15 I 334.0 × 10⁷ 39 5.0 × 10⁷ 293-rtTA 33 2.0 × 10⁸ 39 2.0 × 10³ 293-rtTA-PS-7NI 33 1.5 × 10⁸ 39 3.2 × 10⁸ 293-rtTA-PS-7 I 33 8.0 × 10⁷ 39 5.8 × 10⁷

Cells were infected at multiplicity of 2 plaque-forming unit (p.f.u) percell. 2-3 days later at 39° C. or 5 days later at 32° C., cells wereharvested, frozen-thawed three times, and subsequent extracts weretitrated. Results of a typical experiment are presented here. Titerswere determined as p.f.u. on 293 cells at 33° C. NI: non-induced, I:induced expressions. Experiments carried out at 33° C. were included ascontrols.

While Ad2ts1 yielded respectively 5×10³ p.f.u. and 2×10³ p.f.u. at 39°C. in 293-tTA and 293-rtTA cell lines, complementation was evidenced bythe obtention of titers similar to that of the dl309 mutant in proteaseexpressing cell lines. Induction had the effect of slightly decreasingtiters, but surprisingly, basal expression of the gene from293-tTA-PS-15 and 293-rtTA-PS-7 was sufficient to complement the Ad2ts1mutant. There was no difference between tTA and rtTA complementing celllines.

To further demonstrate the biological activity of cell lines and tocharacterize novel Ad5 mutants, one-step growth curves in 293-tTA/rtTAand 293-tTA/rtTA-PS cell lines were generated (Table 4).

TABLE 4 Yield of Ad5CMVLacZ-CMVGFP-ΔPS and of Ad5-ΔPS with correspondingcontrols from One-Step growth curves in different 293-derived celllines. Virus Cell line Virus Titer Ad5CMVLacZ-CMVGFP-ΔPS 293-tTA/rtTA<10⁴ 293-rtTA-PS-7 NI 10⁸ 293-rtTA-PS-7 I 6.0 10⁷ 293-tTA-PS-15 M 1.410⁸ 293-tTA-PS-15 I 5.0 10⁷ Ad5CMVLaCZ-CMVGFP 293-tTA/rtTA 6.3 10⁸293-rtTA-PS-7 NI 6.5 10⁸ 293-rtTA-PS-7 I 1.2 10⁸ 293-tTA-PS-15 NI 5.010⁸ 293-tTA-PS-15 I 10⁸ Ad5-ΔPS 293-tTA/rtTA <10⁴ 293-rtTA-PS-7 NI 1.410⁸ 293-rtTA-PS-7 I 7.0 10⁷ 293-tTA-PS-15 NI 1.5 10⁸ 293-tTA-PS-15 I 5.210⁷ Ad5 293-tTA/rtTA 6.0 10⁸ 293-rtTA-PS-7 NI 6.5 10⁸ 293-rtTA-PS-7 I1.0 10⁸ 293-tTA-PS-15 NI 6.5 10⁸ 293-tTA-PS-15 I 1.0 10⁸

Cells were infected at a multiplicity of 2 plaque-forming unit (p.f.u)per cell. 2-3 days later, cells were harvested, washed three times inPBS, frozen-thawed three times, and subsequent extracts were titrated.Results of a typical experiment are presented here. Titers weredetermined as p.f u. on 293 cells. NI: non-induced, I: inducedexpression.

Biological activity was also demonstrated by the ability of the293-rtTA-PS-7 clone to generate protease deleted mutantsAd5CMVLacZ-CMVGFP-ΔPS and Ad5-ΔPS after transfection of recombinant DNA.As expected, while the protease deleted mutants were unable to grow in293-rtTA, complementation by cell lines allowed for the restoration ofviral titers close to those of the controls (which are exactly the sameviruses as the mutants, except for the presence of the protease gene).It is noteworthy that as for Ad2ts1, basal protease expression from bothtTA and rtTA complementing cell lines is sufficient to complement theprotease deleted mutants. It is also noteworthy that in the inducedstate of expression of the protease, viral yields were slightlydecreased.

Growth Rate of 293-PS Cell Lines

Visual examination of 293-PS cell lines showed that after induction ofexpression cells did not displayed a significantly different phenotype.To further study the effect of induction on cell lines, viability ofcells was measured by counting living cells, either induced or not,after trypan blue staining every day from D0 to D5. For 293-rtTA-PScells, there was no difference between induced or non-induced cells. Forone clone of 293-tTA-PS (clone 2), results are represented in FIG. 4. Itcan be seen that the expression of the protease had no significantdeleterious effect on cells growth. It is clear as well that as noeffect could be evidenced during a period (24-48 hrs) compatible withthe production of a recombinant adenovirus mutant, these cell lines willbe useful for generation and expansion of protease-deprived mutants.Expression of the gene did not show a toxic effect, but rather a slightcell growth impairment: when maintained in the induced state of proteaseexpression, cells growed slower. Given that overexpression of theprotease could slightly impair cell growth as well as reducing viralyields, controlling its expression with a regulatable promoter wasparamount both for obtaining the best protease complementing cell linesas well as for insuring maximal production of protease deleted AdV.

Stability of 293-PS Cell Lines

To check the stability of selected clones, cell lines maintained during2 months without selection drug were assayed for the expression of theGFP and of the protease. Both proteins were expressed at levels similarto that of early passage cells as determined by respectively flowcytometry and immunoblot analyses (data not shown). No change in drugsusceptibility was noticed after 2 months passages and neither didprotease expression levels were modified.

Transfectability of 293 PS Cell Lines

Clones 293-tTA-PS-15 and 293-rtTA-PS-7 were analyzed for the ability tosupport the production of viral plaques after transfection withAdCMV-LacZ DNA. Both clones yielded as many viral plaques asrespectively parental 293-tTA and 293-rtTA cells. 293PS cell lines werethus very efficiently transfected and were subsequently used for thegeneration of protease-deleted mutants.

Effect of Adenovirus Infection on Protein Expression

To study the effect of the expression of IVa2 products (Lutz et al., J.Virol., 70, 1396-1405, (1996)) on the MLP enhancer that is included inour construction, cell lines were infected in triplicate at a MOI of 1p.f.u. and GFP expression was followed in induced and non-induced cells.No significant difference could be evidenced between both batches.

Generation of Protease-Deleted Mutants

Plasmids clonings are summarized in FIG. 2. For the construction ofprotease-deleted mutants of Ad5, an extension sufficient for homologousrecombination was first introduced in pDE3 plasmid by ligation of the6145 bp fragment resulting from the RsrII/XhoI digestion of the Ad5genome into the unique sites SalI and XhoI. To clone this insert andgenerate pDE3-ext plasmid, RsrII (from the insert) and SalI (from pDE3)were first T4 DNA polymerase repaired. Protease deletion was engineeredby PCR to synthesize a 171 bp upstream fragment (forward primer:gtcgacCATGGACGAGCCCACCCTTCT, SEQ ID NO: 1 reverse primer:ggatccGGCGGCAGCTGTTGTTGATGT) SEQ ID NO: 2, and a 2448 downstreamfragment (forward primer: agatctAAATAATGTACTAGAGACACT, SEQ ID NO: 3reverse primer: ctcgagTTCCACCAACACTCCAGAGTG) SEQ ID NO: 4 (Restrictionsites added for cloning purposes are shown in lower case.) Thesefragments were cloned in the pDE3 plasmid in the SalI and XhoI sites ofthe plasmid, using the BamHI/BglII ligation compatibility. TheSfiI/BamHI fragment from this plasmid was subcloned into pSL1190 plasmid(Pharmacia) and sequenced. It was subsequently cloned into pDE3-extplasmid in the same sites, generating pDE3-ext-ΔPS plasmid. To generateprotease-deleted Ad mutants, recombination into E. coli was chosen (FIG.3). An E1/E3 deleted mutant: Ad5CMVLacZ-CMVGFP-ΔPS was constructed inplasmid by cotransfection in E. coli of the NdeI/XhoI fragment frompDE3-ext-ΔPS with pAdEasy1-βgal-GFP SgfI digested. A mutant deleted onlyfor the protease (Ad5-ΔPS) was generated in the same manner from pTG3602plasmid. Seven micrograms of plasmid DNA PacI digested from bothpAdEasy1-βgal-GFP and pTG3602-ΔPS were transfected in 293-PS-rtTA-7 cellline clone to generate recombinant protease-deleted mutants. The sameamount of recombinant linearized plasmid DNAs were also transfected in293 and 293-rtTA cells as controls. As expected, this experiment yieldedno viral plaques. After 10-14 days viral plaques were observed in293-PS-rtTA. As recombinant adenoviruses have been generated in E.coli,no further cloning of plaques was required (He et al, 1998; Chartier etal, 1996). The whole monolayer was scraped and virus was released fromcells by freeze-thaw cycles. All viral plaques of recombinant virusdisplayed no phenotypic differences from that of wild-type virus.

Ability of Ad5-ΔPS Mutant not Deleted for E1 to Perform a Single Roundof Replication in Non-complementing Cell Lines.

To demonstrate the ability of the mutant deleted for the protease andnot for E1 (Ad5-ΔPS) to perform a single round of replication innon-complementing cell lines, A549 cells were inoculated with wild-type,Ad5-ΔPS, AdΔE1.E3, and Ad5CMVLacZ-CMVGFP-ΔPS viruses. Comparison ofviral protein production (FIG. 6) and of viral yields (FIG. 7) of thedifferent viruses show that only the mutant deleted for the protease andnot for E1 is able of undergoing a single round of replication innon-complementing cells.

The following cell line was deposited with the American Type CultureCollection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209on Dec. 3, 1998 and assigned the following accession numbers:

293 rtTA.PS.7 cell line: ATCC CRL-12595

The following additional biological materials were also deposited underthe Budapest Treaty by applicant with the ATCC on Feb. 17, 1999 and weretested and found viable on May 4, 1999. The accession numbers andIdentification of the deposited materials are as follows.

Human Adenovirus Type 5, Ad5ΔPS VR-2640

Human Adenovirus Type 5, Ad5CMVlacZ_CMVGFP_ΔPS VR-2641

Although various particular embodiments of the present invention havebeen described herein before, for purposes of illustration, it would beapparent to those skilled in the art that numerous variations may bemade thereto without departing from the spirit and scope of theinvention, as defined in the appended claims.

4 1 27 DNA Artificial Sequence Description of Artificial Sequence Primer1 gtcgaccatg gacgagccca cccttct 27 2 27 DNA Artificial SequenceDescription of Artificial Sequence Primer 2 ggatccggcg gcagctgttgttgatgt 27 3 27 DNA Artificial Sequence Description of ArtificialSequence Primer 3 agatctaaat aatgtactag agacact 27 4 27 DNA ArtificialSequence Description of Artificial Sequence Primer 4 ctcgagttccaccaacactc cagagtg 27

What is claimed is:
 1. An adenovirus mutant deleted only for the gene of adenovirus protease.
 2. An adenovirus mutant according to claim 1, further comprising at least one exogenous gene.
 3. An adenovirus mutant according to claim 2, wherein said exogenous gene is expressed from an expression cassette comprising a regulatable promoter.
 4. An adenovirus mutant according to claim 3, wherein said regulatable promoter is a tetracycline derivative responsive promoter.
 5. An adenovirus mutant according to claim 4, wherein said tetracycline derivative responsive promoter is an inducible promoter.
 6. An adenovirus mutant according to claim 5, wherein said tetracycline derivative inducible promoter is the rtTA-dependent minimal promoter.
 7. An adenovirus mutant according to claim 4, wherein said tetracycline derivative responsive promoter is a repressible promoter.
 8. An adenovirus mutant according to claim 7, wherein said tetracycline derivative repressible promoter is the tTA-dependent minimal promoter.
 9. An adenovirus mutant deleted for the gene of adenovirus protease and having the gene cloned in another part of the adenoviral genome.
 10. An adenovirus mutant according to claim 9, wherein the protease gene is cloned in E1 coding region of the genome.
 11. An adenovirus mutant according to claim 10, wherein the protease gene is part of an expression cassette.
 12. An adenovirus mutant according to claim 11, wherein said expression cassette further comprises an exogenous gene.
 13. An adenovirus mutant according to claim 12, wherein said expression cassette is a dicistronic expression cassette.
 14. A method of generating an adenovirus mutant deleted for the gene of endogenous adenovirus protease and having the gene cloned in E1 coding region of adenoviral genome, said method comprising: providing a first piece of adenoviral DNA consisting of an adenoviral genome deleted for the adenovirus protease gene and for the E1 region, providing a second piece of adenoviral DNA which does not contain the E1 region and is capable of recombination in vivo with the first piece of adenoviral DNA, said second piece of DNA having E1 coding region of the adenoviral genome replaced with an expression cassette capable of expressing the gene of adenovirus protease, cotransfecting cells capable of hosting adenovirus with the first and the second piece of adenoviral DNA, incubating said cells, and harvesting recombinant adenovirus.
 15. A method according to claim 14, wherein the expression cassette further comprises an exogenous gene.
 16. A method according to claim 15, wherein the expression cassette is a dicistronic expression cassette.
 17. A method according to claim 14, wherein the cells capable of hosting adenovirus are 293 cell line derivatives. 